Microindentation of fresh soft biological tissue: A rapid tissue sectioning and mounting protocol

Microindentation of fresh biological tissues is necessary for the creation of 3D biomimetic models that accurately represent the native extracellular matrix microenvironment. However, tissue must first be precisely sectioned into slices. Challenges exist in the preparation of fresh tissue slices, as they can tear easily and must be processed rapidly in order to mitigate tissue degradation. In this study, we propose an optimised mounting condition for microindentation and demonstrate that embedding tissue in a mixture of 2.5% agarose and 1.5% gelatin is the most favourable method of tissue slice mounting for microindentation. This protocol allows for rapid processing of fresh biological tissue and is applicable to a variety of tissue types.


GUIDELINES
Process the tissue immediately on arrival to the laboratory.
Maintain tissue hydration in 1X PBS throughout the experiment.

Note
Other brands for consumables are available

SAFETY WARNINGS
Wear appropriate PPE for the duration of the experiment i.e. lab coat, disposable gloves and safety glasses).
Refer to the appropriate safety data sheet for the reagents and chemicals for correct handling and storage.
Dispose of all sharps in a dedicated sharps bin.
Dispose of used reagents in the appropriate waste bins.

ETHICS STATEMENT
The study is approved by the institutional review board and is conducted in accordance with ethical guidelines

Preparation of Phosphate Buffered Saline (PBS)
Preparation of 2.5% (wt/vol) agarose and 1.5% (wt/vol) gelati… tissue.Heat the bottle in the microwave until the agarose and gelatin have dissolved, stopping the microwave every 10-15 seconds to mix the solution with a spatula spoon to ensure the agarose and gelatin are fully dissolved.Once ready, place the bottle in a water bath between 34-37 °C until required for embedding.Do not leave the embedding solution for longer than 00:30:00 , as the embedding solution will solidify after this time point.

Note
Concentration of embedding solution may need to be doubled for certain tissue types such as human intestine and porcine testes which have higher amounts of collagen content. 3 The Compresstome VF-210-0Z (Precisionary Instruments, Massachusetts USA) is set up according to the manufacturing guidelines and is shown in Figure 2a.Briefly, this vibratome uses controllable oscillations and speed to slice through tissue without the use of freezing or fixation of the fresh biological tissue.The oscillation frequency and speed settings are 14 Hz and 14 mm/s respectively.
The razor blade is prepared with scissors as per the user manual and attached to the blade holder using superglue (Figure 1g).The chilled 1X PBS is added to the buffer tray just before sectioning commences (Figure 1b).

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Turn on the water bath and ensure that the temperature is between 34-37 °C

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Prepare 1X PBS for use as a solvent for the embedding solution and as a buffer during tissue sectioning.

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Prepare embedding solution in 40 mL of 1X PBS and heat in a microwave to dissolve.When the agarose and gelatin have dissolved, place in the preheated water bath between 34-37 °C to cool the solution down before embedding.Apply a small amount of superglue to create a thin layer on the surface of the white plunger of the specimen tube (Figure 1c) and carefully adhere one side of the tissue to the base of the white plunger using a tweezers, Figure 3d.Take care to orientate the tissue so that the area of interest is sectioned appropriately.This secures the tissue in place before the embedding solution is added to the tissue.
Once dry, slide the metal tube of the specimen tube (Figure 1d) over the white plunger so that it is level with the height of the tissue, Figure 3d.The tissue is now ready for embedding with the embedding solution. 13 Hold the combined specimen tube containing the tissue sample at a 45 o angle and slowly pipette in the agarose and gelatin embedding solution until the specimen tube is full.Ensure that no air bubbles are in the solution, Figure 3e.

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Remove the chilling block from the -20 °C freezer and slide over the specimen tube for approximately 00:01:00 until a hard gel is formed, Figure 3f.

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Place the specimen into the holder on the buffer tray and secure in place using the thumbscrew.Add the chilled 1X PBS to the buffer tray, Figure 3g.
Attach the blade holder to the vibrating head unit by sliding it onto the axial bar and secure in place 17 As per the manual guidelines of the Compresstome , begin with bigger cutting thicknesses e.g. 1 mm and work in reducing increments to the required thickness in approximately three cuts.

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To mount the tissue for microindentation, gently remove sections from the buffer tray with a paintbrush and place on a drop of liquid agarose and gelatin in a petri dish.The tissue is kept hydrated in 1X PBS at approximately 0-4 °C before indentation to mitigate tissue degradation, Figure 3h,i.
The fresh tissue sections should undergo microindentation within 2 hours of receiving the tissue into the laboratory.Disassemble as per the manual and clean glue off the embedding block and blade mount with acetone.

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Microindentation is performed using the Chiaro Nanoindenter (Optics 11, the Netherlands), Figure 2b.Before each experiment, the probe is calibrated by submerging the cantilever in 1X PBS and using a glass calibration dish.The study is performed at room temperature to prevent measurement error due to temperature drift.The Petri dishes containing the tissue sample are mounted onto an Olympus IX73 microscope, thus allowing visualisation of the tissue during the experiment under the 10X brightfield setting.1X PBS is added, via pipetting, to keep the tissue samples hydrated during the indenting process.Areas of damaged or torn tissue are avoided and indenting is performed away from the edge of the tissue.The probes used to perform this microindentation are within the 0.25 N/m cantilever stiffness and 50 µm radius spherical tip range of probes.An automated 3x3 matrix scan, spaced in 100 μm increments to avoid overlapping, is performed (3).Ultimately, this process allows for the suitable microindentation of fresh soft biological tissue.

30m
Preparation of porcine human tissue for sectioning with vibra… 10mProcedure for the fresh biological tissue sectioning with the C… 40m

Figure 3 Figure 3
Figure 3 Figure 3 Example of tissue preparation, embedding and sectioning for microindentation using human vascular tissue.(a) Human tissue is obtained on the day of surgery.(b) A region of tissue is isolated and cut to approximately 10 mm 2 for microindentation.(c) Samples are hydrated in 1X PBS and placed on ice for approximately 10 minutes prior to embedding.(d) The tissue segment is glued to the white specimen tube and allowed to dry for 1 minute.The metal specimen tube is placed onto the white specimen tube once the glue is dry.(e) The agarose and gelatin embedding solution is added to the specimen tube.(f) The filled specimen tube is rapidly cooled with a chilling block or on ice for approximately 1 minute.(g) The cooled specimen tube is mounted into the vibratome.(h) The tissue is sectioned to the desired thickness.(i) The tissue section is mounted onto the liquid agarose and gelatin embedding solution.The tissue is hydrated in 1X PBS before microindentation (Created with BioRender.com).